In situ protein micro-crystal fabrication by cryo-FIB for electron diffraction

Area of Science:

• Structural Biology
• Biophysics
• Crystallography

Background:

• Micro-electron diffraction (MicroED) is an emerging technique for macromolecular crystallography.
• Conventional X-ray crystallography is unsuitable for micro-/nano-crystals.
• Limited production of suitable crystals and crystal transfer hinder MicroED application.

Purpose of the Study:

• To develop an efficient workflow for preparing suitable crystals for MicroED.
• To expand the applicability of MicroED to a wider range of crystal sizes.
• To bridge the gap between MicroED and X-ray crystallography.

Main Methods:

• In situ on-grid crystallization
• Single-side blotting
• Cryo-focus ion beam (cryo-FIB) fabrication
• Cryo-electron diffraction of crystal cryo-lamella

Main Results:

• A complete workflow for efficient crystal preparation for MicroED was developed.
• The workflow enables MicroED analysis of 2-10 μm macromolecular crystals.
• Successfully solved the 2.5 Å crystal structure of lysozyme from a 10 μm³ micro-crystal.

Conclusions:

• The developed workflow significantly expands the range of crystals suitable for MicroED.
• This method addresses limitations in crystal preparation and transfer for MicroED.
• The workflow effectively bridges the size gap between MicroED and X-ray crystallography.